in vitro reconstitution of the selection, ubiquitination, and membrane extraction of a polytopic ERAD substrate.
in vitro reconstitution of the selection, ubiquitination, and membrane extraction of a polytopic ERAD substrate.
复制标题
多位 ERAD 底物的选择、泛素化和膜提取的体外重建。
DOI：
10.1007/978-1-60327-412-8_21
复制
发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Brodsky,JeffreyL
中科院分区：
文献类型：
--
作者：
Nakatsukasa,Kunio;Brodsky,JeffreyL
真核生物细胞内细胞器的分泌和膜蛋白首先在内质网(ER)合成，然后被运送到它们的最终目的地。内质网含有高浓度的分子伴侣和折叠酶，帮助底物获得其天然构象。然而，蛋白质错误折叠是不可避免的事件，特别是当细胞暴露在压力下，或者在发育或老化过程中。内质网相关降解(ERAD)是从分泌途径中消除错误折叠蛋白的主要机制。分泌和膜蛋白的突变或ERAD机制的腐败与人类疾病有关，这一事实突显了ERAD的重要性。通过对酿酒酵母的遗传分析，已经确定了ERAD中的许多成分，现在看来，这些因子中的大多数在高等真核生物中是保守的。在这一章中，我们描述了一种利用酵母制备的材料，在体外概括泛素化和错误折叠的多孔膜蛋白提取的方法。这些技术为进一步剖析ERAD途径的基本步骤提供了强大的工具。
Secretory and membrane proteins that are destined for intracellular organelles in eukaryotes are first synthesized at the endoplasmic reticulum (ER) and are then delivered to their final destinations. The ER contains high concentrations of molecular chaperones and folding enzymes that assist substrates to acquire their native conformations. However, protein misfolding is an inevitable event especially when cells are exposed to stress or during development or aging. ER-associated degradation (ERAD) is a major mechanism to eliminate misfolded proteins from the secretory pathway. The importance of ERAD is underscored by the fact that mutations in secretory and membrane proteins or corruption of the ERAD machinery have been linked to human diseases. Many components involved in ERAD have been identified by a genetic analysis using the yeastSaccharomyces cerevisiae, and it now appears that most of these factors are conserved in higher eukaryotes. In this chapter, we describe a method to recapitulate the ubiquitination and extraction of misfolded polytopic membrane proteins in vitro using materials prepared from yeast. These techniques provide a powerful tool to further dissect the ERAD pathway into elementary steps.